The present invention relates to the general field of genetic vaccines and relates, in particular, to genetic agents delivered into the skin or mucosal tissues of animals to induce an immune response, and more particularly to genetic vaccines for viral pathogens delivered into skin or mucosal tissues by particle acceleration.
In particular, the present invention relates to the field of genetic vaccines that protect human and non-human vertebrates against infection by viruses of the Filovirus genus (Family Filoviridae). The known filoviruses include the Ebola virus Reston, Ebola virus Sudan, Ebola virus Zaire and Marburg virus (strain Musoke and strain Ravn). Filoviruses are non-segmented, negative stranded enveloped ssRNA viruses having a vertebrate host range. The range of possible invertebrate hosts (such as, but not limited to, arthropods) is not known. A glycoprotein inserted in the viral envelope may mediate virus entry into host cells.
The Marburg virus glycoprotein (170 kD) is a type I transmembrane protein. The carbohydrate structures account for more than 50% of the molecular weight of the protein. The Ebola virus glycoproteins (125 kD) appear to have similar carbohydrate structures to the Marburg glycoproteins, except insofar as the Ebola glycoproteins are terminally sialated.
Marburg and Ebola viruses cause severe hemorrhagic fever in humans and in laboratory primates. Ebola-Zaire strain appears to be more deadly than either the Sudan strain or the Marburg virus. After an incubation period of four to sixteen days, sudden fever, chills, headache, anorexia and myalgia appear. Nausea, vomiting, sore throat, abdominal pain and diarrhea soon follow. Most patients develop severe hemorrhaging between about days five and seven. Death usually occurs between seven and sixteen days.
In patients, antibodies directed primarily against the surface glycoproteins of Marburg and Ebola viruses can be detected as early as ten to fourteen days after infection. However, it is not entirely clear that such antibodies can prevent the overt manifestations of the disease.
The vaccination of individuals to render the vaccinated individuals resistant to the development of infectious disease is one of the oldest forms of preventive care in medicine. Previously, vaccines for viral and bacterial pathogens for pediatric, adult, and veterinary usage were derived directly from the infectious organisms and could be categorized as falling into one of three broad categories: live attenuated, killed, and subunit vaccines. Although the three categories of vaccines differ significantly in their development and mode of actions, the administration of any of these three categories of these vaccines is intended to result in production of specific immunological responses to the pathogen, following one or more inoculations of the vaccine. The resulting immunological responses may or may not completely protect the individual against subsequent infection, but will usually prevent the manifestation of disease symptoms and significantly limit the extent of any subsequent infection.
The techniques of modern molecular biology have enabled a variety of new vaccine strategies to be developed which are in various stages of pre-clinical and clinical development. The intent of these efforts is not only to produce new vaccines for old diseases, but also to yield new vaccines for infectious diseases in which classical vaccine development strategies have so far proven unsuccessful. Notably, the recent identification and spread of immunodeficiency viruses is an example of a pathogen for which classical vaccine development strategies have not yielded effective control to date.
The first broad category of classical vaccine is live attenuated vaccines. A live attenuated vaccine represents a specific strain of the pathogenic virus, or bacterium, which has been altered so as to lose its pathogenicity, but not its ability to infect and replicate in humans. Live attenuated vaccines are regarded as the most effective form of vaccine because they establish a true infection within the individual. The replicating pathogen and its infection of human cells stimulates both humoral and cellular compartments of the immune system as well as long-lasting immunological memory. Thus, live attenuated vaccines for viral and intracellular bacterial infections stimulate the production of neutralizing antibodies, as well as cytotoxic T-lymphocytes (CTLs), usually after only a single inoculation.
The ability of live attenuated vaccines to stimulate the production of CTLs is believed to be an important reason for the comparative effectiveness of live attenuated vaccines. CTLs are recognized as the main component of the immune system responsible for the actual clearing of viral and intracellular bacterial infections. CTLs are triggered by the production of foreign proteins in individual infected cells of the hosts, the infected cells processing the antigen and presenting the specific antigenic determinants on the cell surface for immunological recognition.
The induction of CTL immunity by attenuated vaccines is due to the establishment of an actual, though limited, infection in the host cells including the production of foreign antigens in the individual infected cells. The vaccination process resulting from a live attenuated vaccine also results in the induction of immunological memory, which manifests itself in the prompt expansion of specific CTL clones and antibody-producing plasma cells in the event of future exposure to a pathogenic form of the infectious agent, resulting in the rapid clearing of this infection and practical protection from disease.
An important disadvantage of live attenuated vaccines is that they have an inherent tendency to revert to a new virulent phenotype through random genetic mutation. Although statistically such a reversion is a rare event for attenuated viral vaccines in common use today, such vaccines are administered on such a large scale that occasional reversions are inevitable, and documented cases of vaccine-induced illnesses exist. In addition, complications are sometimes observed when attenuated vaccines lead to the establishment of disseminated infections due to a lowered state of immune system competence in the vaccine recipient. Further limitations on the development of attenuated vaccines are that appropriate attenuated strains can be difficult to identify for some pathogens and that the frequency of mutagenic drift for some pathogens can be so great that the risk associated with reversion are simply unacceptable. A virus for which this latter point is particularly well exemplified is the human immunodeficiency virus (HIV) in which the lack of an appropriate animal model, as well as an incomplete understanding of its pathogenic mechanism, makes the identification and testing of attenuated mutant virus strains effectively impossible. Even if such mutants could be identified, the rapid rate of genetic drift and the tendency of retroviruses, such as HIV, to recombine would likely lead to an unacceptable level of instability in any attenuated phenotype of the virus. Due to these complications, the production of a live attenuated vaccine for certain viruses may be unacceptable, even though this approach efficiently produces the desired cytotoxic cellular immunity and immunological memory.
The second category of vaccines consists of killed and subunit vaccines. These vaccines consist of inactivated whole bacteria or viruses, or their purified components. These vaccines are derived from pathogenic viruses or bacteria which have been inactivated by physical or chemical processing, and either the whole microbial pathogen, or a purified component of the pathogen, is formulated as the vaccine. Vaccines of this category can be made relatively safe, through the inactivation procedure, but there is a trade-off between the extent of inactivation and the extent of the immune system reaction induced in the vaccinated patient. Too much inactivation can result in extensive changes in the conformation of immunological determinants such that subsequent immune responses to the product are not protective. This is best exemplified by clinical evaluation of inactivated measles and respiratory syncytial virus vaccines in the past, which resulted in strong antibody responses which not only failed to neutralize infectious virions, but exacerbated disease upon exposure to infectious virus. On the other extreme, if inactivating procedures are kept at a minimum to preserve immunogenicity, there is significant risk of incorporating infectious material in the vaccine formulation.
The main advantage of killed or subunit vaccines is that they can induce a significant titer of neutralizing antibodies in the vaccinated individual. Killed vaccines are generally more immunogenic than subunit vaccines, in that they elicit responses to multiple antigenic sites on the pathogen. Killed virus or subunit vaccines routinely require multiple inoculations to achieve the appropriate priming and booster responses, but the resultant immunity can be long lasting. These vaccines are particularly effective at preventing disease caused by toxin-producing bacteria, where the mode of protection is a significant titer of toxin neutralizing antibody. The antibody response can last for a significant period or can rapidly rebound upon subsequent infection, due to an anamnestic or secondary response. On the other hand, these vaccines generally fail to produce a cytotoxic cellular immune response, making them less than ideal for preventing viral disease. Since cytotoxic lymphocytes are the primary vehicle for the elimination of viral infections, any vaccine strategy which cannot stimulate cytotoxic cellular immunity is usually the less preferred methodology for a virus disease, thereby resulting in attenuated virus being the usual methodology of choice.
The development of recombinant DNA technology has now made possible the heterologous production of any protein, of a microbial or viral pathogen, or part thereof, to be used as a vaccine. The vaccine constituents thus do not need to be derived from the actual pathogenic organism itself. In theory, for example, viral surface glycoproteins can be produced in eukaryotic expression systems in their native conformation for proper immunogenicity. However, in practice, recombinant viral protein constituents do not universally elicit protecting antibody responses. Further, as with killed vaccines, cellular cytotoxic immune responses are generally not seen after inoculation with a recombinant subunit protein. Thus, while this vaccine strategy offers an effective way of producing large quantities of a safe and potentially immunogenic viral or bacterial protein, such vaccines are capable of yielding only serum antibody responses and thus may not be the best choice for providing protection against viral disease.
The availability of recombinant DNA technology and the developments in immunology have led to the immunological fine mapping of the antigenic determinants of various microbial antigens. It is now theoretically possible, therefore, to develop chemically synthetic vaccines based on short peptides in which each peptide represents a distinct epitope or determinant. Progress has been made in identifying helper T-cell determinants, which are instrumental in driving B-cell or antibody immune responses. The covalent linkage of a helper T-cell peptide to a peptide representing a B-cell epitope, or antibody binding site, can dramatically increase the immunogenicity of the B-cell epitope. Unfortunately, many natural antibody binding sites on viruses are conformation-dependent, or are composed of more than one peptide chain, such that the structure of the epitope on the intact virus becomes difficult to mimic with a synthetic peptide. Thus peptide vaccines do not appear to be the best vehicle for the stimulation of neutralizing antibodies for viral pathogens. On the other hand, there is some preliminary evidence that peptides representing the determinants recognized by cytotoxic T-lymphocytes can induce CTLs, if they are targeted to the membranes of cells bearing Class I Major Histocompatibility Complex (MHC) antigens, via coupling to a lipophilic moiety. These experimental peptide vaccines appear safe and inexpensive, but have some difficulty in mimicking complex three dimensional protein structures, although there is some evidence that they can be coaxed into eliciting cytotoxic immunity in experimental animals.
Another new recombinant technique which has been proposed for vaccines is to create live recombinant vaccines representing non-pathogenic viruses, such as a vaccinia virus or adenovirus, in which a segment of the viral genome has been replaced with a gene encoding a viral antigen from a heterologous, pathogenic virus. Research has indicated that infection of experimental animals with such a recombinant virus leads to the production of a variety of viral proteins, including the heterologous protein. The end result is usually a cytotoxic cellular immune response to the heterologous protein caused by its production after inoculation. Often a detectable antibody response is seen as well. Live recombinant viruses are, therefore, similar to attenuated viruses in their mode of action and result in immune responses, but do not exhibit the tendency to revert to a more virulent phenotype. On the other hand, the strategy is not without disadvantage in that vaccinia virus and adenovirus, though non-pathogenic, can still induce pathogenic infections at a low frequency. Thus it would not be indicated for use with immune-compromised individuals, due to the possibility of a catastrophic disseminated infection. In addition, the ability of these vaccines to induce immunity to a heterologous protein may be compromised by pre-existing immunity to the carrier virus, thus preventing a successful infection with the recombinant virus, and thereby preventing production of the heterologous protein.
In summary, all of the vaccine strategies described above possess unique advantages and disadvantages which limit their usefulness against various infectious agents. Several strategies employ non-replicating antigens. While these strategies can be used for the induction of serum antibodies which may be neutralizing, such vaccines require multiple inoculations and do not produce cytotoxic immunity. For viral diseases, attenuated viruses are regarded as the most effective, due to their ability to produce potent cytotoxic immunity and lasting immunological memory. However, safe attenuated vaccines cannot be developed for all viral pathogens.
It is therefore desirable that vaccines be developed which are capable of producing cytotoxic immunity, immunological memory, and humoral (circulating) antibodies, without having any unacceptable risk of pathogenicity, or mutation, or recombination of the virus in the vaccinated individual.
To date, no pharmaceutical or immunological methods exist for preventing filovirus infections or for intervening after infection. Vaccination with viral antigens or inactivated whole virus vaccines have been ineffective in protecting against challenge with live virus. No antiviral drug has been effective, even in vitro. Thus, no specific treatments exist for the diseases caused by filoviruses.
Published PCT patent application Nos. PCT/US93/02394 and PCT/US93/02338, both incorporated herein by reference, relate to genetic immunizations for viruses using viral DNA.